Topography and recognition imaging atomic force microscope and method of operation

ABSTRACT

A recognition force microscope for detecting interactions between a probe and a sensed agent on a scanned surface and methods for its operation are provided. The microscope includes a scanning probe having a tip that is sensitive to a property of the scanned surface, and the probe is adapted to oscillate with a low mechanical Q factor. Operation of the microscope includes recording the displacement of the probe tip as a function of time and simultaneously recording both topographic images and the spatial location of interactions between said probe and one or more sensed agents on the surface.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. ProvisionalApplication Serial No. 60/423,222, filed Nov. 1, 2002.

BACKGROUND OF THE INVENTION

[0002] The present invention relates to an atomic force microscope andmethods of operating that microscope to provide both topographic andrecognition imaging, and more particularly to an atomic force microscopefor detecting interactions between a probe and a sensed agent on thesurface of a substrate to provide simultaneous topographic andrecognition images.

[0003] It has long been recognized that the atomic force microscope canbe made to be sensitive to specific chemical interactions between aprobe tip and a surface. For example, Lee et al., “Sensing discretestreptavidin-biotin interactions with atomic force microscopy,” Langmuir10:354-357 (1994), demonstrated specific binding between biotin andstreptavidin using chemically modified cantilever probes. Anotherexample of a specific interaction between chemically reactive groups isgiven by Kienberger et al., “Static and dynamical properties of singlepoly (ethylene glycol) molecules investigated by force microscopy,”Single Molecules 1:123-128 (2000).

[0004] A method for attaching antibodies to a scanned probe has beendescribed by Hinterdorfer et al., “Detection and localization ofindividual antibody-antigen recognition events by atomic forcemicroscopy,” Proc. Natl. Acad. Sci. (USA) 93: 3477-3481 (1996); “Forcespectroscopy of anti-body-antigen recognition measured by scanning forcemicroscopy.” Biophys. J. 74:186 (1998); and “A mechanistic study of thedissociation of individual antibody-antigen pairs by atomic forcemicroscopy,” Nanobiology 4:39-50 (1998). This method has been used tocharacterize interactions between several antibody-antigen pairs. Themethod has also been used to characterize interactions between adhesiveproteins (Baumgartner, Hinterdorfer et al., “Cadherin interaction probedby atomic force microscopy.” Proc. Natl. Acad. Sci. USA 97 (2000)) andbetween ligands and transporter molecules embedded in native proteinmembranes.

[0005] Those skilled in the art will recognize that the technique isquite general and applicable to any set of materials that bind to oneanother—receptors with their corresponding proteins, drugs with theirligands, and antibodies with antigens. Thus, the chemical bonded to theprobe may be termed the sensing agent, while the chemical recognized ona sample surface may be termed the sensed agent.

[0006] In the prior art methods described above, single moleculeinteraction forces are measured with chemically modified cantileverprobe tips in molecular recognition force spectroscopy (MRFS)experiments using so-called force distance cycles: Highly selectiveligands (preferentially one per tip) are covalently attached to thetip-end as shown in FIG. 1A. Referring to FIG. 1A, a cantilever probetip 1 is modified with a specific reagent (such as ethanolamine oraminopropyltriethoxysilane) to place reactive groups (such as amines) onthe surface of the probe 2. An amine reactive group 3, attached to oneend of a flexible 8-nm long crosslinker 4, tethers the crosslinker(which may be polyethylene glycol (PEG) to the probe. A second reactivegroup (for sulfurs in this case, pyridine dithioproprionate (PDP.)) 5reacts with the thiolated surface of the sensing agent, as carried outwith the thiolating agent N-succinimidyl 3-(acetylthio) proprionate(SATP) 6, for example.

[0007] In this way, the sensing agent, in this case an antibody 7, isheld on the end of the crosslinker 4. This arrangement has the advantagethat the tethered sensing agent (the antibody 7) is free to move to theextent that the crosslinker 4 is flexible, thereby allowing the sensingagent to align with its target sensed agent (in this case the specificantigen for the antibody) on the surface being probed. Binding of thesensing agent 7 on the tip 1 with a specific sensed agent on the surfacecan be observed in the force-distance curve obtained as the tip isscanned towards the surface and retracted as shown in FIG. 1B.

[0008] The tip 1 is moved towards the surface of a sample, which leadsto the formation of a single receptor-ligand bond between the tetheredantibody and specific antigen on the sample surface. The force curve onapproach shows no sign of this bond formation (“trace” 8). However, onretraction of the tip, a characteristic curve is observed (“retrace” 9)showing an increasing attractive force as the crosslinker 4 is stretcheduntil the bond is broken when the retraced distance equals the almostfully extended length of the crosslinker at 10. The characteristic shapein the retrace reflects the viscoelastic properties of the crosslinker 4by which the antibody 7 is tethered to the tip 1.

[0009] In the prior art described thus far, the surface must be probedby carrying out force-distance curves at every point of potentialinterest. In Elings et al, U.S. Pat. No. 5,519,212, the patentees statethat the interaction between an antibody and an antigen can be detectedby changes in the oscillation of a vibrated tip, although there is nodescription of how this may be accomplished. Raab et al provided thefirst practical demonstration of antibody-antigen recognition in ascanned image (Raab, Han et al., “Antibody recognition imaging by forcemicroscopy,” Nature Biotech. 901-905 (1999)). In this work, a dynamicforce microscope was operated in MACMODE (a trademark of MolecularImaging Corp.), a mode in which the tip motion is controlled by anapplied magnetic field. This mode of operation is described in greaterdetail in Lindsay, U.S. Pat. Nos. 5,515,719 and 5,513,518 and in Han,Lindsay et al., “A magnetically-driven oscillating probe microscope foroperation in liquids,” Appl. Phys. Letts. 69:4111-4114 (1996).

[0010] Raab, Han et al. describe that the tip was driven intooscillation with an amplitude similar to the length of the crosslinker(4 in FIG. 1A) used to tether an antibody to the end of the tip. Theantibody was antilysozyme and the antigen on the substrate was lysozyme.Referring to FIG. 2A, when a bare tip 21 is used to image the lysozyme22, images such as those in FIG. 2C are obtained. When a modified tip 23with antilysozyme 24 is attached (as shown in FIG. 2B), the images ofthe lysozyme are greatly broadened and increased in apparent height asshown in the image in FIG. 2E. The difference in the appearance of theimages is illustrated by the line scans in FIG. 2D. The trace 25 overlysozyme taken with the bare tip 21 is narrower and lower than the trace26 taken with the antibody tip 23, reflecting the attachment of antibodyto antigen and subsequent stretching of the crosslinker as described byRaab, Han et al.

[0011] Receptor-ligand recognition is monitored by an enhanced reductionof the oscillation amplitude as a result of antibody-antigen binding.These binding signals are visible as bright and wide dots in therecognition image and reflect the position of ligand binding-sites withnanometer (nm) lateral accuracy. The drawback to this methodology isthat the antibody-enzyme binding signals in the recognition image areinterfered with by signals owing to the topographic features of theenzyme. Topography and recognition images can only be recorded bycomparing a pair of images taken with bare and antibody-conjugated tips,respectively, and are, therefore, not obtained at the same time.

[0012] An increase in the speed of molecular recognition imaging ishighly desirable, not just for increased effectiveness of microscopy,but also because a rapid molecular recognition method would enable verymany small titer wells to be examined for binding affinity, opening aroute for rapid drug screening. Accordingly, the need exists in the artto provide an atomic force microscope and method of operation thatprovides separate yet simultaneous topography and recognition images. Aneed also exists for a method for the rapid quantitative measurement ofmolecular binding with high spatial resolution.

SUMMARY OF THE INVENTION

[0013] The present invention meets these needs by providing an atomicforce microscope and method of operating it that provides separate yetsimultaneous topography and recognition images as well as rapidquantitative measurement of molecular interactions with high spatialresolution. The present invention may be useful in providing highspatial resolution of many physical, chemical, and biologicalinteractions on both hard and soft surfaces.

[0014] In accordance with one aspect of the present invention, arecognition force microscope for detecting interactions between a probeand a sensed agent on a scanned surface is provided and includes ascanning probe having a tip that is sensitive to a property of asurface, with the probe adapted to oscillate with a low mechanical Qfactor. By “Q factor,” we mean the quality factor of a cantilever probe,where Q=f₁/Δf₁, where f₁ is the first resonance frequency of thecantilever and Δf₁ is the full width of the resonance peak athalf-maximum. By “low mechanical Q factor” we mean a Q factor of greaterthan zero and equal to or less than about 20. The Q factor of thecantilever is determined by the stiffness of the cantilever and theviscosity of the medium in which it oscillates, and also, to someextent, by the geometry of the cantilever. A Q factor of about equal toor less than 20 is typical of what might be measured for cantilevershaving a stiffness of a few Newtons per meter oscillated in water. Thisis typical of the conditions used for imaging biological materials withan atomic force microscope (AFM).

[0015] The microscope also includes means for recording the displacementof the probe tip as a function of time and means for recording bothtopographical data and recognition data, i.e., the spatial location ofinteractions between the probe and sensed agents on the surface. In oneembodiment, the means for recording the displacement of the probe tip asa function of time include a source of radiation such as a laser that isdirected at the probe, a position sensitive detector that detectsradiation reflecting off of the surface of the probe, and a controllerthat processes the detected radiation. In one embodiment, the means forrecording both the topographical and recognition data includesprocessing circuitry that generates separate topographical andrecognition signals. In one embodiment, the amplitudes of the respectiveupward and downward swings (displacements) of the probe tip are recordedand are used to determine both topographic data and recognition data toidentify the spatial location of interaction sites between the probe tipand sensed agents on a sample surface.

[0016] Preferably, the probe tip is sensitized with a sensing agent thatbinds specifically to the sensed agent. In one preferred embodiment ofthe invention, the sensing agent is an antibody and the sensed agent isan antigen. The sensing agent, such as for example an antibody, may betethered to the probe tip by a flexible crosslinker (i.e., a chemicalagent that binds the sensing agent to the probe tip). Other sensingagent/sensed agent pairs may be utilized. For example, many ligand andreceptor pairs are known in the art. Many drugs, toxins, haptens,transmitters; and agonists are known to interact with receptormolecules. Sense and antisense DNA and DNA-RNA proteins also interact.However, the apparatus and methods of the present invention are notlimited to molecular binding or bonding but also include other chemicaland physical interactions such as, for example, electrostatic chargeinteractions and hydrophobic/hydrophilic interactions. Thus, the“sensing agent” on the probe tip may include electrical and/or chemicalmodifications to the tip as well as tethering of molecules to the tip.

[0017] In one embodiment, a time varying magnetic field is used toexcite the probe into motion using a magnetic material that forms atleast a portion of the probe. In a preferred form, the topographic andrecognition data signals that are detected and recorded are separated byan electronic circuit that includes means for determining the averagevalue of the displacement of the probe (using a deflection signalgenerated from the position sensitive detector) on a time scale that issufficiently long compared to changes caused by topography or bindingevents such that those events are separately recognized and measured.The electronic circuit also includes means for using the average valueof the displacement of the probe to determine the downward amplitude ofthe probe from the difference between the average value and the value ofthe downward displacement. In a preferred form, these means include adigital signal processor operating using a recognition-imagingalgorithm.

[0018] The electronic circuit also includes means for controlling theheight of the probe. In a preferred form the means for controlling theheight of the probe includes a piezoelectrically driven scanning elementin conjunction with a controller. Thus, topography is determined usingthe downward value of the probe tip displacement. The electronic circuitalso includes means for determining the value of the upward displacementof the probe from the measured amplitude and the average value of thedisplacement to generate a signal corresponding to interactions betweena sensing agent and a sensed agent on the surface being scanned. In apreferred form, the means for determining these values includes adigital signal processor operating using a recognition-imagingalgorithm.

[0019] In another embodiment of the invention, the topographic andrecognition signals are separated by an electronic circuit that includesmeans for digitizing the recorded deflection of the probe tip andcomputing means for determining the average value of the displacement ofthe probe tip on a time scale that is sufficiently long compared tochanges caused by topography or binding events such that those eventsare separately recognized and measured. In a preferred form, thedigitizing means includes one or more A/D converters. The electroniccircuit also includes means for using the average value of thedisplacement of the probe to determine the downward amplitude from thedifference between the average value and the value of downwarddisplacement. In a preferred form, the means for determining thesevalues includes a digital signal processor operating using arecognition-imaging algorithm.

[0020] The electronic circuit also includes means for controlling theheight of the probe to determine the topography of the sample using thevalue of downward displacement and means for determining the value ofthe upward displacement from the upward amplitude and the average valueof displacement to generate a signal corresponding to interactionsbetween a sensing agent on the probe tip and a sensed agent on thesurface being scanned.

[0021] In another embodiment of the invention, the probe tipdisplacement measured as a function of time is used to determine thespatial location of recognition events by comparison to a predicted orrecorded displacement pattern generated for the case when there is norecognition.

[0022] The present invention also provides a method of operating anatomic force microscope including scanning a probe oscillating with alow mechanical Q factor and that is sensitive to a property of asurface, recording the displacement of the probe tip as a function oftime, and simultaneously recording both topographical images and thespatial location of interactions between the probe and sensed agents onthe surface of a sample. Preferably, the method uses the extent of theupward displacement of the probe tip to measure interactions between theprobe tip and the sample surface. The height of the probe tip above thesample surface is controlled by using either the extent of the downwarddisplacement of the probe tip (i.e., bottom amplitude), the overallamplitude of the probe tip (i.e., the sum of the upper and loweramplitudes of the tip divided by two), or the average deflection signal(i.e., the difference between the upper and lower amplitudes of thetip).

[0023] In another embodiment of the invention, a method for screeningreagents for binding to a particular target molecule is provided andincludes attaching the target molecule to the tip of a probe, scanningat least one candidate reagent with an oscillated force-sensing probeoperating with a low mechanical Q factor, using the extent of thedownward displacement of the probe to control the height of the probeabove the sample surface, and using the extent of the upwarddisplacement to measure interactions between the target molecule and thecandidate reagent. Preferably, the method is used to screen for multiplecandidate reagents sequentially. For example, the candidate reagents maybe placed in an array and sampled sequentially.

[0024] In yet another embodiment of the invention, a method of screeningligands for binding to a particular target on a cell surface is providedand includes attaching the ligand to the tip of a probe, scanning a cellsurface with an oscillated force-sensing probe operated with a lowmechanical Q factor, using the extent of the downward displacement tocontrol the height of the probe above the sample surface, and using theextent of the upward displacement to measure interactions between atleast one target molecule on the cell surface and the candidate ligand.

[0025] Accordingly, it is a feature of the present invention to providean atomic force microscope and method of operating it that providesseparate and simultaneous topography and recognition images as well asrapid quantitative measurement of molecular binding with high spatialresolution. This and other features and advantages of the invention willbecome apparent from the following detailed description, theaccompanying drawings, and the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Reference will now be made by way of example to the drawings inwhich:

[0027]FIG. 1A is a schematic illustration of a chemically-modifiedcantilever probe tip;

[0028]FIG. 1B is a graph of a force versus distance curve showing bondformation and breaking when the chemically-modified cantilever probe tipof FIG. 1A is moved toward and away from a sample surface;

[0029]FIG. 2A is a schematic illustration of an unmodified probe tipinteracting with a lysozyme that produces an image as shown in FIG. 2C;FIG. 2B is a schematic illustration of a chemically-modified probe tipinteracting with a lysozyme that produces an image as shown in FIG. 2E;FIG. 2D provides a comparison of the height and width of images producedby the probe tips illustrated in FIGS. 2A and 2B;

[0030] FIGS. 3A-3C illustrate characteristic waveforms for oscillationof a free probe tip (A), a probe tip that contacts a surface (B), and aprobe tip that both contacts a surface and becomes bound thereto (C);

[0031]FIGS. 4A and 4B illustrate plots of probe tip displacement as theprobe tip is scanned across a surface, with FIG. 4A being a plot for abare tip, and FIG. 4B being a plot for a tip having a sensing agentattached thereto;

[0032]FIG. 5 is a schematic illustration of one embodiment of anelectronic circuit that separates topography and recognition signals;

[0033]FIGS. 6A and 6B are simultaneous topography and recognitionimages, respectively, that are produced when a probe tip having asensing agent tethered thereto is used to image a surface containingsensed agents;

[0034]FIGS. 7A and 7B are simultaneous topography and adhesion images,respectively, that are produced when a bare probe tip is used to image asurface containing sensed agents;

[0035]FIG. 8 is a schematic illustration of a digital electronic circuitfor one embodiment of the invention that separates topography andrecognition images;

[0036]FIG. 9 is a flow chart depicting an algorithm used in oneembodiment of the invention for recognition imaging;

[0037]FIG. 10 is an illustration of recognition and topography signalsobtained from distortion of a probe tip oscillation waveform;

[0038]FIG. 11 is a schematic illustration of a scanning probe microscopeuseful in the practice of one or more embodiments of the presentinvention;

[0039] FIGS. 12A-12C are charts depicting a raw deflection signal from asurface scan having upper and lower peaks, with FIG. 12A depicting aprobe tip encountering a change in surface topography, FIG. 12Bdepicting a deflection event as a functionalized probe tip encountersand binds with a sensed agent on a surface, and FIG. 12C depicting theabsolute value of the peak and peak differential signals derived fromFIG. 12B;

[0040]FIG. 13 is a schematic illustration of an embodiment of a peakdifferential detector that provides a recognition signal;

[0041]FIG. 14 is a schematic illustration of an analog electroniccircuit for another embodiment of the invention that separate topographyand recognition images; and

[0042]FIGS. 15A and 15B are plots representing probe tip displacement asa probe tip is scanned across hard and soft surfaces, respectively.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] The present invention provides a novel apparatus and method for amolecular force recognition microscopy (MFRM) operational mode that iscapable of recording recognition and topography images simultaneouslyand independently. When an atomic force microscope cantilever with a lowmechanical Q factor is oscillated in a fluid, the amplitude at the topof the cantilever swing is not related to the amplitude at the bottom ofthat swing when the tip intermittently contacts a surface. Suchindependence is nearly complete for cantilevers operated with a Q factorof greater than zero but less than about 2 to 3, but is still evident tosome extent with cantilevers with a Q factor of less than about 20.Cantilevers with low Q-factors (approximately 1 in liquid) that aredriven at frequencies below resonance contain recognition andtopographic information that are well separated. The time resolvedoscillation signals are specially processed before they are fed backinto a microscope feedback loop as will be explained in greater detailbelow.

[0044] As shown schematically in FIG. 11, an atomic force microscope(AFM) useful in the practice of the present invention includes acantilever 28, having a film 29 comprising a magnetic ormagnetostrictive material on the top surface thereof. Cantilever 28includes a probe tip 30 extending from the bottom surface thereof towardsample 32. The probe tip 30 is scanned over the surface 34 of sample 32by a piezoelectric scanner 36. Deflections of cantilever 28 are detectedby directing a focused beam of radiation 38 a from, for example, a laser40 off of the reflective surface of film 29 to form a reflected beam 38b. Position sensitive detector 42 senses the angular position of beam 38b.

[0045] A solenoid 64 located in proximity to cantilever 28 is driven byan AC signal from an oscillator 66 as more fully set forth in Lindsay,U.S. Pat. No. 6,121,611, the disclosure of which is hereby incorporatedby reference. The resulting alternating magnetic field causes cantilever28 to oscillate, producing both upward and downward displacement of theprobe tip. This oscillating motion appears as an AC signal component inthe signal that is detected by position sensitive detector 42. Thissignal, which includes both upward and downward swing peak values, isfed to a processing circuit 50 that generates both topographic andrecognition image signals as will be explained in greater detail below.

[0046] The voltage from the position detector 42 used to sense bendingof the AFM cantilever (detector voltage) and is plotted as a function oftime for three specific cases in FIG. 3. FIG. 3A shows the signal whenthe cantilever probe does not contact the surface at any time during anoscillation. The waveform is sinusoidal with a peak displacement valueU₊ and a lower displacement value U⁻. The average value, U_(M), lieshalf way between U₊ and U⁻. It is not, in general, zero, because thedetector signal is DC coupled for preferred embodiments of themicroscope of the present invention. When the probe tip contacts thesurface (FIG. 3B), the lower amplitude is changed to a different value,U⁻′. The voltage corresponding to the upper extent of the cantileverswing (displacement), U₊, is unchanged. The new average value is U_(M)′and the new peak-to-peak value is (U₊−U⁻′). It is this peak-to-peakvalue (or a proportional quantity, the root mean square value) that isused to control the microscope as topographical images are obtained.

[0047] Thus, automatic feedback control through a servo loop inconjunction with the microscope controller acts to maintain the waveformshown in FIG. 3B as the probe is scanned over the surface, with theheight changes needed to accomplish this being recorded as thetopographical image. For example, as shown in FIG. 11, height errorsignal 57 is processed by controller 60 that results in a Z heightcorrection signal being sent through high voltage amplifier 62 topiezoelectric scanner 36. If the servo control of height were to bereleased as the probe encountered a binding site (on an otherwise flatsurface), the waveform shown in FIG. 3C would be obtained. Because thesurface was assumed to be flat, U⁻′ continues at its previous value.

[0048] However, if the overall amplitude of the oscillation iscomparable to the crosslinker length (4 in FIG. 1A), then the amplitudeof the upward swing is reduced if the tethered sensing agent binds asensed agent on the surface. Thus, the positive peak voltage is changedto a new value, U₊′. Thus, changes in U₊′ relative to a baseline signalbefore binding, U_(M)′, reflect interactions between the sensing agenton the tip and the sensed agent on the surface, but not the surfacetopography if the cantilever Q is low enough, i.e., less than about 20,and preferably less than about 3. Correspondingly, changes in the lowervoltage, U⁻′ relative to U_(M)′, reflect the topography, but notinteractions between the sensing agent and the sensed agent.

[0049] This sensitivity is illustrated, by way of example, in recordingsof the deflection of the oscillating tip as it is scanned over a lineacross a surface covered with sparsely distributed single distributedenzyme molecules that are specific antigens for antilysozyme on theprobe. To illustrate the effect, scans were made with the servo controlof height switched off. The results are shown in FIG. 4.

[0050] Scanned with a bare tip (no tethered antibody, e.g. 21 in FIG.2A), the deflection signal recorded as the probe tip is scanned in asweep across the sample is shown in FIG. 4A. Because the microscopeservo loop is disabled, the lower amplitude (U⁻′, 41) fluctuates as theprobe scans over enzymes on an otherwise flat surface. However, becausethe tip has a low mechanical Q factor, the upper amplitude, U₊′ 42remains unaffected. When, however, an antibody functionalized probe tipis scanned over the same surface (FIG. 4B) both U⁻′ (43) and U₊′ (44)fluctuate.

[0051] The data in FIG. 4 are presented in compressed form (20,000 fullperiods of oscillation shown in total) so that only the minima (lowerborder line of envelope) and maxima (upper border line of envelope) ofthe respective oscillation periods are visible. The surface contact onlyinfluences the downward deflections (minima) of the oscillations.Therefore, enzymes on the surface are solely detected by the oscillationminima (bumps in the lower line of the envelope) and the maxima remainconstant within the thermal noise of the cantilever deflection.

[0052] If, however, the same sample is scanned with anantibody-containing tip, bumps are also visible in the upper borderline44 (FIG. 4B). Binding of the antibody on the tip to the enzyme on thesurface reduces the upward deflection, because the tip is physicallyconnected to the surface via the flexible crosslinker (4, FIG. 1A). Thetip oscillation amplitude is ideally chosen to be just slightly smallerthan the extended crosslinker length, so that both the antibody remainsbound while passing a binding site and the reduction of the upwarddeflection is of sufficient significance compared to the thermal noiseto be measured. Because the spring constant of the crosslinker isnonlinearly increasing with the tip-surface distance, the binding forceis only sensed close to full extension of the crosslinker (given at themaxima of the oscillation period). Therefore, the recognition signalsare well separated from the topographic signals arising from thesurface, both in space (Δz of approximately 5 nanometer) and time (halfoscillation period of approximately 0.1 millisecond).

[0053] There are a number of possible ways to implement the practice ofthe present invention, and those skilled in this art will recognize thatthe invention is not limited to the specific electrical circuitrydescribed herein. In one embodiment of the present invention, topographyand recognition images are obtained simultaneously using circuit 50illustrated in FIG. 5. The signal from the detector 42 is passed to apeak detector 52 and maxima (U₊) and minima (U⁻) of each sinusoidalcantilever deflection period are detected, filtered, and amplified. DCoffset signals 53 and 54 are used to compensate for thermal drifts. Thesignals are chosen so as to be close to U_(M)′, but adjusted during thescan as needed to compensate for slow drifts in U_(M)′. Values for U₊and U⁻ are fed into the AFM controller 56 with U⁻ driving the feedbackloop 55 to record a height (topography) image 57 in a normal manner andU₊ providing the data for constructing a recognition image 58 bydisplaying U₊ as a function of position on the surface. In this way,topography and recognition images are obtained both simultaneously andindependently.

[0054] Results of the operation of the circuit of FIG. 5 with a tetheredantibody are shown in FIG. 6. The topography image 57 is shown in FIG.6A, and it shows many enzymes located on the surface with dimensionssimilar to those obtained in images that use the peak-to-peak amplitudeas the control parameter (e.g., FIG. 2C). The image in FIG. 6A wasobtained with a tethered antibody. The recognition image 58 is shown inFIG. 6B. Dark spots are seen for some of the molecules, corresponding toa dip in U₊ as antibody and antigen bind. For a given cluster ofmolecules 61 only a fraction show simultaneous recognition imagingevents 62. However, the lateral positions of enzymes recorded in thetopography are spatially correlated with the recognition signals of therecognition image.

[0055] This method of one embodiment of the present invention works evenin cases where a flexible tether is not used, and this is illustrated byrecognition images of ferritin, a highly positively charged protein. Itwas sensed with a negatively charged AFM cantilever probe tip (but whichwas not specifically chemically modified, in contrast to the examplesdescribed previously). When the AFM tip touches a ferritin moleculetip-protein adhesion due to electrostatic attraction leads to decreaseof the upward deflection. An almost perfect match between positions ofproteins in the topography image 57 (FIG. 7A) and localization of chargeinteraction in the adhesion image 58 (FIG. 7B) is visible. In this case,the amplitude of oscillation is chosen to be so low that the tip isnever completely free from the protein on the upward swing, despite thelack of a flexible crosslinker.

[0056] The method of one embodiment of the invention is sensitive tochemical adhesion between the probe and the surface even in the absenceof a tether. This is because the upper swing of the cantilever is dampedto some extent by events that dissipate energy on the lower swing of thecantilever (because the Q is on the order of 1 or a little more). Thus,local points of high adhesion lead to reduction in the amplitude of thelower swing. The overall damping (both upper and lower parts of theswing) leads to an apparent high point in the topography as thecontroller pulls the tip away from the substrate to restore theoscillation amplitude. By monitoring the recognition signalsimultaneously with the topography signal, embodiments of the presentinvention permit discrimination between topographical features that arereal (i.e., caused by a real change of height) and those that are aconsequence of local changes in adhesion (i.e., change in surfacechemistry with no change of height).

[0057] These alternative modes of operation demonstrates that thedescribed embodiments of the present invention have a broad range ofapplications as a high resolution sensing apparatus for the correlationbetween topographic structures and localization of specific interactionin general. However, the embodiments described above operate best forflat surfaces and when the drift of the instrument is not too large.This is because the average DC value of the raw displacement signal doesvary even when the peak-to-peak amplitude is under servo control as aresult of drift and errors in the servo when rough terrain is scanned.

[0058] These disadvantages are overcome in another embodiment of thepresent invention that is shown schematically in FIG. 8. The raw signals(A and B represent the signals from the upper and lower segments of thephotodetector, respectively, from the diode detector 42 segments) are DCcoupled into analog to digital converters 82 and 83 and fed to a digitalsignal processor 84 where the deflection signal is formed by calculatingthe difference of the segment voltages divided by their sum as shown at85. The height control signal 87, topography image data stream 88 and asubsequent algorithm 86, further described below, obtains recognitionimage data stream 89. Alternatively, the deflection waveform could beobtained by analog methods and then converted to a digital form forsubsequent processing.

[0059] The recognition-imaging algorithm 86 is described by theflowchart in FIG. 9. In a first step 92, the following quantities arecalculated in DSP 84 (FIG. 8) using the digitized detector waveform 91obtained as described above:

[0060] U₊, the sliding average peak positive value of the deflectionsignal where the signal is polarized so that positive voltagescorrespond to upward swings of the cantilever. The time period of thesliding average is τ.

[0061] U⁻, the lowest sliding time average value of the deflectionsignal. The time period of the sliding average is τ.

[0062] U_(M), the mean averaged value of voltage, given by a slidingtime average of (U₊−U⁻)/2. The time period of this sliding average isT>τ.

[0063] τ is chosen to permit a rapid response of the servo whileminimizing noise. For example, if one line scan of 512 pixels isaccumulated in 1 second, then τ=1/512s=0.0195 seconds. A tip vibrationfrequency of 5 kHz, then results in 0.0195/0.0002=10 cycles averaged foreach value of U₊ and U⁻. T is chosen so as to give a static value forU_(M) in the absence of recognition events. Because the servo feedbackcontrol loop maintains U⁻, then, in the absence of recognition events,U_(M) should be a constant. However, mechanical and thermal drifts causethe average position of the probe to change slowly. Therefore, U_(M)must be averaged over a period that is small compared to the total scantine for an image, but larger than the time spent by the probe when itis engaged in a recognition event. A suitable time is about {fraction(1/20)}^(th) of a line scan time, or 0.05 seconds in this case. Thistime may be user-adjustable, being shortened when the image areacontains a high density of binding sites.

[0064] The values of U⁻(t) and U₊(t) are stored each τ seconds intemporary memory locations 93 where the two previous values U⁻(t−τ) andU₊(t−τ) are also stored. U_(M)(t) is updated every τ seconds (calculatedas a sliding average over T seconds) and the current value U_(M)(t) andthe prior value, U_(M)(t−τ), stored as well. The quantity U₊(t−τ)−U₊(t)is formed only when a scan is first started (94). If this quantity isless than zero (so that the upper voltage is rising with time), the scanwas started on a recognition site. In this case, the microscope servo isengaged using the R=0 pathway (97, 98) and the scan continued untilU₊reaches a stable value (U₊(t−τ)−U₊(t)≧0) after which the signal issubsequently tested for U₊(t−τ)−U₊(t)>0 (96). If the signal falls(U₊(t−τ)−U₊(t)>0), then a recognition event has occurred and controlbranches to 900. If not, an error signal is calculated from thedifference of a set point value (user controlled), SP andU_(M)(t)−U⁻(t), the downward value of the deflection. This is used tocontrol the height of the probe in a conventional manner and thus todetermine the sample topography (and height control signal) via theusual feedback circuit 99 as is well known in the art. This feedbackpath applies only if no recognition event is in progress. In this casethe flag R is zero so the U_(M)(t)−U⁻(t) signal is connected to theservo via the switch 98.

[0065] When U₊(t−τ)−U₊(t)>0 occurs, flagging a recognition event, therecognition flag is set to R=1, the value of the mean signal is set tothe value just prior to recognition, i.e., S_(M)=U_(M)(t−τ) so thatS_(M) is not updated like U_(M) but corresponds to the value of U_(M)just prior to recognition. The value of the peak amplitude just atrecognition is stored as UP=U₊(t₀), where t₀ represents the start time.Finally, a new peak positive value, Sp is calculated using S_(M)according to S_(p)(t)=U₊(t)−S_(M) as shown in 900. While the upperamplitude remains below the value at the onset of sensing(S_(p)(t)−UP<0) control continues in the recognition mode (901).S_(p)(t) is recorded in a separate image buffer to generate arecognition display like those shown in FIGS. 6B and 7B. The heightservo is now controlled from the signal SP−(S_(M)−U−(t)) at 904 andpassed on to the servo 99 via the switch 905. When the upper amplitudereaches its value at the start of recognition, S_(p)(t)−UP>0, thecontrol mode returns to normal (R=0) at 902 and U_(M) is calculated as asliding average again starting from the stored value S_(M).

[0066] It will be recognized that other algorithms may be devised toaccomplish the same results. For example, the servo feedback control canbe continued on one path only, using U_(M), so long as T is much greaterthan the time spent in a recognition event. This would result in someerror in the topography signal, but it would correctly identifyrecognition events.

[0067] Another, more computationally intensive, but faster method is tocompare each cycle of the oscillation amplitude to a best fit sine waveas illustrated in FIG. 10. Here the solid line shows the instantaneousvalue of the displacement data stream 101, while the dots correspond tothe best-fit sine wave 102. The peak difference between the upper valueof the data and the upper part of the fit (Δ U₊) at 103 gives theamplitude decrease owing to recognition binding and can be plotted asthe recognition signal. The peak difference between the lower value ofthe data and the lower part of the fit (Δ U⁻) at 104 gives the amplitudedecrease owing to topography and can be used to control the height servoloop.

[0068] In another embodiment of the invention, as shown in FIGS. 12A-12Cand 13, the microscope is operated in the normal manner to generatetopographic signals. An included circuit in the microscope views changesin the upper portion of the raw deflection signal as a function of timeto provide spatial recognition of binding events and a recognition imagesimultaneously with topographic signals.

[0069] Referring now to FIGS. 12A-12C and 13, the servo feedback controlloop is operated from the AC coupled deflection signal in a normalmanner to provide the best image. By way of example, it is assumed thatthe microscope is engaged with ±5 nm amplitude when in contact with thesurface (greater than this before engagement). FIG. 12A shows the rawdeflection signal as a 1 nm high object spanning 40 to 60 ms on the timeaxis is scanned. At 201 the surface diminishes the lower extent of thecantilever probe swing. At 202 the servo control activates, pulls thesample away from the probe tip, and restores the full amplitude of theoscillating probe. At 203 the surface of the scanned object falls away,increasing the lower amplitude of the oscillation. At 204 the servocontrol again activates and pushes the sample toward the probe tip againto reach the engage amplitude.

[0070] By way of example, FIG. 12B shows the sequence of events as anantibody tip functionalized with a tethered antibody on a 3 nm tetherpasses over an antigenic objection a scanned surface spanning 40 to 60ms in the time axis. At 205 the antibody binds, causing the peakamplitude on the top swing of the probe to reduce. At 206, the servocontrol activates and pulls the sample away from the probe tip. Thiscauses the negative peak amplitude to become more negative. The apparentdisplacement recorded in FIG. 12B has nothing to do with translation ofthe sample, an event that causes negligible bending change in thecantilever. Rather, the amplitude loss comes from the top portion of theswing, but the servo control restores the bottom portion to its previousnegative amplitude. At 207 the probe tip encounters the sample, reducingthe lower swing amplitude. At 208, the servo control activates again,pulling the sample further away from the probe tip. At 209, the probetip reaches the edge of the object, increasing the lower amplitude ofthe deflection signal. At 210 the servo control activates again andpushing the sample toward the probe tip to restore the lower amplitude.At 211 the antibody unbinds from the probe tip, and at 212 the servocontrol acts to pull the sample away from the probe tip to compensate.

[0071]FIG. 12C shows the absolute value of the peak amplitude. It isdepressed when the probe is over the antigen on the scanned surface, buthas spikes in it where the servo control is reacting. The peak signalprovides useful recognition of the binding and unbinding events.However, the signal should be corrected for drift. One way to correctfor drift (given a real time signal) is to take the real timepoint-to-point derivative of the peak signal, shown as the dashed linein FIG. 12C. This signal is predominantly negative as the object (e.g.,antigen) first binds, is zero, and then becomes predominantly positive.Thus, an image formed from this signal would show the binding event asringed by a black ring on the approaching perimeter and a white ring onthe receding perimeter.

[0072] An embodiment of a detector circuit implementing the abovedetection scheme is shown in FIG. 13. Referring to FIG. 13, the DCcoupled deflection signal 301 is fed into a square wave generator 302comprising an AC coupling and a Schmidt trigger. This generates adigital clock 303 synchronized to the deflection signal. The RS(reset/set) flip flop divides the result by two as shown. The outputs ofthe flip-flop, Q and {overscore (Q)}, drive analog switches 304, 305 inseries with two peak detectors 306, 307, respectively, so that the peakpositive value on alternate cycles is held on the two peak detectors.The falling edge of the {overscore (Q)} output generates a short delaypulse 308 (small compared to a cycle) and operates the sample and hold309 to store the difference between the peak values, P_(n+1)−P_(n). Asecond delay pulse 310 resets the peak detectors. An RC time constant isused to provide signal averaging. It should be user switchable and coverfrom a single cycle to a few cycles so that servo-relateddiscontinuities are smoothed (e.g., a time constant from 0.1 to 10 ms in5 steps).

[0073] In yet another embodiment of the invention, a recognition imagingcircuit provides both topographical and recognition imaging signals foran MFRM. As illustrated in FIG. 14, the raw deflection signal from thedetector (e.g., 42 in FIG. 11) is passed through a low pass filter 101to a subtraction circuit 102 where the output is the difference betweenthe low-pass filtered signal and the unfiltered signal. A feature ofthis circuit is to remove the offset of the oscillating tip-deflectionsignal caused by drift and other factors. Thus the time constant (RC) ofthe low pass filter is set to a value comparable to the time taken toscan a line in the microscope (for example, from about 0.1 to about 1.0seconds).

[0074] The raw deflection signal includes a sine wave, corresponding tothe probe tip oscillation, superimposed on a fluctuating DC levelassociated with local changes in the environment. At the output side oflow pass filter 101 and subtraction circuit 102, the signal comprisesonly the sine wave signal, oscillating about zero volts. Thus, thecircuit ‘filters’ out the slow component of the change in the rawdeflection signal but leaves the absolute DC value of the signal intact.Using a capacitor in the circuit at this point would distort thewaveform.

[0075] This filtered or steadied signal is now passed to a peak detectorcircuit 103. A peak detector circuit records the peak level reached bythe input voltage. However, it is desirable to record the change in peakvoltage from cycle to cycle, because this change corresponds to therecognition signal. Accordingly, the steadied signal is fed to acomparator 105 to generate a digital signal from the sine wave inputfrom the steadied signal. The threshold voltage at which the comparatorfires should be set so that circuit noise will not trigger an output.However, if the threshold voltage is set too high, the input sine wavefrom the steadied signal will not trigger the comparator.

[0076] In order to provide recognition imaging, the probe tiposcillation amplitude must be similar to the length of the flexibletether used to hold the antibody (or other recognition element) to thetip. This, in turn dictates the amplitude of the steadied deflectionsignal arriving at the comparator 105, given that the electronic gainsprior to this stage are fixed for each selected sample to be scanned. Wehave found that setting the threshold voltage to trigger comparator 105to be from about 10 to about 70% of the positive part of the steadiedsignal, a usable recognition-imaging signal is provided. Thus, where theoscillating part of the steadied deflection signal is 1 volt peak topeak at the input to comparator 105, then the threshold voltage shouldbe set at 0.10 to 0.7 v, and preferably 0.25 to 0.35V.

[0077] The digital waveforms coming from comparator 105 drive a dualD-type flip flop circuit 106. This arrangement causes the output Q1 topulse when the deflection signal passes the threshold voltage going upand the output Q2 to pulse when the deflection signal passes thethreshold voltage going down. These signals (Q2 is shown on line 107 andQ1 is shown on line 108) are fed to a reset signal on the positive peakdetector 103 and to the sample control input of a sample and holdcircuit 104. Thus, on one cycle, the output of positive peak detector103 is sampled and then held by sample and hold circuit 104. On the nextcycle, positive peak detector 103 is reset. It is then sampled again onthe cycle after that.

[0078] The result is that the value of the positive peak on every othercycle is displayed on the output of sample and hold circuit 104. This isthe recognition signal. The sample and reset operations generate acertain amount of high frequency noise, so the recognition signal maythen fed through a low pass filter 109 to generate a final recognitionsignal 110 that is then fed to an auxiliary display channel of themicroscope controller (not shown). Typically, this second low passfilter 109 is set to pass frequencies corresponding to the duration ofthe recognition signal, but to cut off higher frequencies. Thus, if therecognition signal extends over 1% of a line scan of the image, and theline scan speed is 0.1 seconds, the filter would be set to pass signalsbelow about 1.0 kHz (1/(0.1×0.001)).

[0079] A parallel circuit with a negative peak detector 111, sample andhold circuit 112 and filter 113 generates a signal corresponding to thebottom amplitude of the deflection signal. The bottom amplitude could beused to control the operation of microscope. However, in practice betterperformance may be obtained if the raw deflection signal is used tocontrol the microscope's sample height servo feedback loop in the normalmanner used for AFM imaging. Alternatively, the overall amplitude of thesignal may be used as the control for the feedback servo loop. In thosecases, bottom peak signal 114 may be used as a diagnostic tool. Innormal operation, bottom peak signal 114 should mirror the ‘deflection’or ‘error’ signal obtained by displaying the amplitude of the deflectionsignal, a signal commonly available on the microscope controller. Properoperation of the microscope may be monitored by checking that the bottompeak signal output 114 and the microscope error signal follow oneanother.

[0080] The embodiments of the invention described above have beenillustrated in terms of the amplitude changes that occur on recognitionsites attached to hard surfaces. The present invention is also useful inpracticing MRFM on soft surfaces. FIG. 15A summarizes a plot of theoscillation amplitude as an oscillated probe is approached towards ahard surface (solid line) and then retracted after a recognition event(e.g. antibody binding—dashed line). The dashed horizontal line (4)shows the set-point amplitude reduction at which the microscopeoperates.

[0081] Prior to a binding event, the probe tip rests at an averagedistance Z1 from the surface of a sample. A binding event will cause adrop in the overall oscillation amplitude because of the reduction ofthe top part of the swing. However, the steep increase of overallamplitude on pushing away from a hard surface (the portion of the curveshown at 1) compensates for the loss of amplitude due to binding withjust a small motion of the probe away from the surface. Thus, the newoperating point of the microscope (distance Z2) is established at apoint (e.g., Z2) that is well within the range where the antibodyremains bound (i.e., before the bond is broken as shown at 3).

[0082] In the case where the sample comprises a soft surface, such as acell membrane or soft polymer as shown in FIG. 15B, the increase ofamplitude with distance moved away from the surface is much smaller(i.e., the slope of the curve in region 5). Once again, dashed line 6shows the set point, and the operating distance in the absence ofbinding is Z1. Now, when binding occurs, the probe tip must move muchfurther from the soft surface to recover from a given degree of dampingcaused by, for example, antibody binding. In those instances where thesample surface is much softer than the antibody tether, the pulling awaycontinues for large distances until the antibody-antigen bond is brokenentirely as shown at 7 before a new stable operating point isestablished. At this point, the probe tip is again advanced to thesurface to return to the set point amplitude 6, but re-binding causesthe cycle to repeat.

[0083] Thus, in the case of a soft surface, a recognition event ischaracterized by a series of spikes in amplitude. Because the probe tipposition is unstable during a recognition event, it is no longerpossible to recover a true topography signal. Nonetheless, the largeamplitude series of spikes serve as the basis for detecting arecognition event by monitoring the deflection signal as the sample isscanned.

[0084] It will be recognized that the present invention enables a veryrapid screening process for molecular recognition imaging. The target ofinterest is tethered to the AFM cantilever probe tip using the methodswell known in the art (e.g., using the heterobifunctional linkers astaught by Hinterdorfer et al (1996)). The prospective ligands arearranged in microtiter wells (which could be printed by nanolithographicdip-pen methods) as a close-packed array on an imaging substrate. Thearray is imaged, recording the recognition signal as a marker of bindingstrength, while simultaneously recording the topography as an index sothat recognition signals can be associated with particular wells.

[0085] Alternately, the method can be used to screen the efficacy withwhich particular ligands bind sites on diseased cells. The ligand istethered to the cantilever probe tip and cell surfaces are scanned.Comparison between the recognition signals on healthy and normal cellswill identify the discrimination of the ligand for diseased cells inaddition to locating the specific location of binding sites on thediseased cells.

[0086] The invention having being described with reference to preferredembodiments, it will be apparent that the same may be varied in manyways. Such variations are not to be regarded as a departure from thespirit and scope of the invention, and all such modifications as wouldbe obvious to one skilled in the art were intended to be included withinthe scope of the invention.

What is claimed is:
 1. A recognition force microscope for detectinginteractions between a probe and a sensed agent on a scanned surface,comprising: a scanning probe having a tip that is sensitive to aproperty of said surface, said probe adapted to oscillate with a lowmechanical Q factor; means for recording the displacement of said probetip as a function of time; and means for recording both topographicimages and the spatial location of interactions between said probe andone or more sensed agents on said surface.
 2. A microscope as claimed inclaim 1 in which said means for recording the displacement of said probetip as a function of time comprise a source of radiation directed atsaid probe, a position sensitive detector that detects radiationreflecting off of said probe, and a controller that processes thedetected radiation.
 3. A microscope as claimed in claim 1 in which saidmeans for recording both topographical images and the spatial locationof binding events between said probe and sensed agents comprisesprocessing circuitry that generates separate topographical andrecognition signals.
 4. A microscope as claimed in claim 1 in which theamplitude of the upward swing and the amplitude of the downward swing ofsaid probe tip are measured and recorded.
 5. A microscope as claimed inclaim 1 where the Q factor is 20 or less.
 6. A microscope as claimed inclaim 1 wherein the probe tip is sensitized with a sensing agent thatbinds specifically to the sensed agent.
 7. A microscope as claimed inclaim 6 wherein said sensing agent is an antibody.
 8. A microscope asclaimed in claim 7 wherein said antibody is tethered by a flexiblecrosslinker.
 9. A microscope as claimed in claim 6 where said sensingagent is tethered by a flexible crosslinker.
 10. A microscope as claimedin claim 1 in which said probe includes a magnetic material, and saidmicroscope further includes a time varying magnetic field adapted toexcite said probe into motion.
 11. A microscope as claimed in claim 4including an electronic circuit for separating the topographic andrecognition signals, said circuit comprising, means for determining theaverage value of the displacement of said probe on a time scale that issufficiently long compared to changes caused by topography or bindingevents such that such events are separately recognized; means for usingsaid average value of said displacement to determine the downwardamplitude of said probe from the difference between said average valueand the value of the downward displacement; means for controlling theheight of said probe, thereby determining the topography using saidvalue of the downward displacement of said probe; and means fordetermining the value of the upward displacement of the probe from theupward amplitude and said average value of said displacement to generatea signal corresponding to interactions between said probe and saidsensed agent on the surface being scanned.
 12. A microscope as claimedin claim 4 where the topographic images and the spatial location ofbinding events are separated by an electronic circuit comprising, meansfor digitizing the recorded displacement of said probe tip; means fordetermining the average value of the displacement of said probe on atime scale that is sufficiently long compared to changes caused bytopography or binding events such that such events are separatelyrecognized; means for using said average value of said displacement todetermine the downward amplitude of said probe from the differencebetween said average value and the value of the downward displacement;means for controlling the height of said probe, thereby determining thetopography using said value of the downward displacement of said probe;and means for determining the value of the upward displacement from theupward amplitude and said average value of said displacement to generatea signal corresponding to interactions between said probe and saidsensed agent on the surface being scanned.
 13. A recognition forcemicroscope for detecting interactions between a probe and a sensed agenton a scanned surface comprising, a scanning probe having a tip that hasbeen sensitized to a property of said surface, said probe adapted tooscillate with a low mechanical Q factor; a source of radiation directedat said probe; a position sensitive detector for detecting radiationreflected from said probe; a processor for processing signals from saiddetector to determine both topographic images and the spatial locationof interactions between said probe and sensed agents on said surface.14. A microscope as claimed in claim 13 in which the amplitude of theupward swing and the amplitude of the downward swing of said probe tipare measured and recorded.
 15. A microscope as claimed in claim 13 wherethe Q factor is 20 or less.
 16. A microscope as claimed in claim 13wherein the probe tip is sensitized with a sensing agent that bindsspecifically to the sensed agent.
 17. A microscope as claimed in claim16 wherein said sensing agent is an antibody.
 18. A microscope asclaimed in claim 17 wherein said antibody is tethered by a flexiblecrosslinker.
 19. A microscope as claimed in claim 16 where said sensingagent is tethered by a flexible crosslinker.
 20. A method of operatingah atomic force microscope comprising, scanning a probe having a tipthat is sensitive to a property of a surface of a sample over saidsurface while oscillating said probe with a low mechanical Q factor;recording the displacement of said probe tip as a function of time; andsimultaneously recording both topographic images and the spatiallocation of interactions between the probe and sensed agents on saidsurface.
 21. A method as claimed in claim 20 in which the Q factor is 20or less.
 22. A method as claimed in claim 20 including using the extentof the upward displacement of said probe tip to measure interactionsbetween said probe tip and the sample surface.
 23. A method as claimedin claim 20 including using the extent of the downward displacement ofsaid probe tip to control the height of said probe tip above the samplesurface.
 24. A method as claimed in claim 20 including using the overallamplitude of said probe tip to control the height of said probe tipabove the sample surface.
 25. A method as claimed in claim 20 includingusing the average deflection signal to control the height of said probetip above the sample surface.
 26. A method of screening reagents forbinding to a particular target molecule comprising, attaching the targetmolecule to the tip of a probe and scanning the surface of a samplecontaining at least one candidate reagent while oscillating said probetip with a low mechanical Q factor; using the extent of the downwarddisplacement of said probe tip to control the height of the probe abovethe sample surface; and using the extent of the upward displacement ofsaid probe tip to measure interactions between the target molecule andthe candidate reagent.
 27. A method as claimed in claim 26 in which theQ factor is 20 or less.
 28. A method as claimed in claim 26 includingusing the tip displacement as a function of time to determine thespatial location of recognition events by comparison to a predicted orrecorded displacement pattern generated for the case when there is norecognition.
 29. A method as claimed in claim 26 in which candidatereagents are arranged in microtiter wells arrayed on a substrate.
 30. Amethod as claimed in claim 29 including simultaneously recording bothtopographic images and the spatial location of interactions between thetarget molecule and the candidate reagents such that recognition eventsare associated with specific wells.
 31. A method of screening ligandsfor binding to a particular target on a cell surface comprising,attaching the ligand to the tip of a probe and scanning said cellsurface while oscillating said probe tip with a low mechanical Q factor;and using the extent of the downward displacement of said probe tip tocontrol the height of the probe above the sample surface; and using theextent of the upward displacement to measure interactions between thetarget on the cell surface and the ligand.
 32. A method as claimed inclaim 31 in which the Q factor is 20 or less.
 33. A method as claimed inclaim 31 including using the tip displacement as a function of time todetermine the spatial location of recognition events by comparison to apredicted or recorded displacement pattern generated for the case whenthere is no recognition.
 34. A microscope for detecting surfacetopography simultaneously with probe-surface interactions, saidmicroscope comprising: means for causing the probe to sample distance tovary with time rapidly as compared to the time to move the probe overthe surface, means for detecting the peak amplitude of probe motion ateach cycle of variation of probe to sample distance, means for recordingthe surface topography based on the average amplitude of motion of theprobe in relation to said surface, and means for recording variations inthe peak amplitude of probe motion as a function of the probe positionover the surface.